CN112980928A

CN112980928A - Stem loop primer-assisted isothermal nucleic acid amplification method

Info

Publication number: CN112980928A
Application number: CN202110361417.0A
Authority: CN
Inventors: 罗光成; 易婷婷; 郭晓兰; 张国元
Original assignee: Affiliated Hospital of North Sichuan Medical College
Current assignee: Affiliated Hospital of North Sichuan Medical College
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-06-18
Anticipated expiration: 2041-04-02
Also published as: CN112980928B

Abstract

A stem loop primer assisted isothermal nucleic acid amplification technology is characterized by comprising the following steps: a. SPA primer design: firstly, designing two common primers, namely a forward primer FP and a reverse primer BP, aiming at a template; b. after designing a common primer, adding stem-loop sequences at the 5' ends of FP and BP to form a pair of stem-loop primers, namely a forward stem-loop primer SFP and a reverse stem-loop primer SBP; c. preparation of SPA primer mixed solution: the SPA amplification primer is formed by mixing common primers FP and BP and stem-loop primers SFP and SBP; d. SPA buffer preparation 2 XSAEmix: weighing potassium chloride, ammonium sulfate, magnesium sulfate heptahydrate, betaine, Tris-HCl, dNTP, 1.0 mL of 100-time diluted SYBR green I and Tween-20, fully dissolving and fixing the volume, and subpackaging each tube at-20 ℃ for storage; e. preparation of SPA reaction liquid: the SPA reaction is a 10-50 muL system. The method has the advantages of simple operation, rapidness, sensitivity, economy, practicability and the like, can realize the rapid amplification and detection of nucleic acid, and is particularly suitable for field detection and rapid screening.

Description

Stem loop primer-assisted isothermal nucleic acid amplification method

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a stem loop primer-assisted isothermal nucleic acid amplification method.

Background

The nucleic acid amplification technology is one of the most widely used examination technologies in life science research, and plays a very important role in the fields of basic research, clinical disease diagnosis, infectious disease prevention and control, food safety, environmental monitoring and the like. There are two main categories of nucleic acid amplification techniques: one is a temperature cycling based Polymerase Chain Reaction (PCR), and the other is a constant reaction temperature based Isothermal Amplification Technology (IAT).

PCR is the most widely used nucleic acid amplification technology at present, the amplification principle is simpler, and only one pair of primers and one DNA polymerase are needed to realize the amplification reaction. Therefore, the PCR technology has been widely used in basic research and clinical practice. However, with the development of medical environments, doctors and patients have made demands on nucleic acid detection faster, more accurate, more economical, and the like. The disadvantages of PCR have also become very prominent. Because, PCR techniques require expensive temperature cycling to effect the amplification reaction, and require specialized technicians to effect nucleic acid detection. This makes PCR difficult to use for point-of-care testing, field testing, home testing, and testing in economically laggard areas. Since rapid detection and field detection are important for basic diagnosis and infectious disease prevention and control, isothermal amplification technology, which is simple, rapid, sensitive, efficient, economical and practical, has received much attention in recent years.

Isothermal amplification technology is a technology for amplifying and detecting nucleic acid at constant temperature, and has the characteristics of simplicity, rapidness and high efficiency. Over the past two decades, more than ten isothermal amplification techniques have been invented, including loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), helicase-dependent isothermal amplification (HDA), Rolling Circle Amplification (RCA), nucleic acid sequence-based isothermal amplification (NASBA), Strand Displacement Amplification (SDA), and the like. Unfortunately, these isothermal amplification techniques have several disadvantages and thus none have been widely used in clinical practice.

LAMP is a relatively widely used isothermal amplification technology, and has the typical defects of complicated primer design, large primer quantity and poor amplification specificity. Efficient LAMP amplification requires 6 primers designed for eight sections of a template, which not only makes primer design difficult, but also further causes stronger background amplification and poorer specificity of LAMP technology. The primer is numerous, so that primer dimer is easy to cause, and the mismatching probability of the primer and background nucleic acid is increased, thereby causing non-specific amplification.

RPA is also a widely used isothermal amplification technique in recent years, but at least 5 proteins, two primers and one probe (SauDNA polymerase, RecA recombinase, single-stranded DNA binding protein, T4 UvsY protein and creatine kinase, pre-primer, post-primer and fluorescent probe labeled THF) are required to achieve RPA amplification. Therefore, the RPA reagent is complex in composition and expensive. HAD required modification of dNTP substrates and nonspecific amplification was evident. RCA can only amplify circular templates. The existing isothermal amplification technologies have some defects. Therefore, the development of a more sensitive, specific, simple, reliable, economical and practical isothermal amplification technology is expected to realize wide clinical application.

The prior art closest to the present invention is the LAMP technology, and the technical principle and the implementation scheme are introduced as follows:

the technical principle of LAMP is as follows:

loop-mediated isothermal amplification (LAMP) was an isothermal nucleic acid amplification technique invented by Notomi et al in 2000. At least 4 primers (whose sequence is derived from 6 regions of the template) are required for the basic version of LAMP to achieve amplification. As shown in the attached figure 1, 6 template regions related to the LAMP primers of the basic edition are respectively as follows: f1, F2, F3, B1, B2 and B3, wherein the 4 amplification primers consisting of the 6 regions are respectively as follows: an anterolateral primer (FIP), a posteromedial primer (BIP), an anterolateral primer (F3), and a posterolateral primer (B3). In addition, in order to increase the reaction speed, a pair of accelerating primers, namely a forward accelerating primer (LF) and a reverse accelerating primer (LB), can be added, namely an accelerating LAMP.

The amplification principle of the basic version of LAMP is shown in the attached FIG. 2: the double-stranded DNA is in a dynamic equilibrium state of melting-renaturation at about 65 ℃, and under the catalysis of Bst DNA polymerase with strand displacement activity (which is a functional basis of isothermal amplification), an inner primer (comprising FIP and BIP) and an outer primer (comprising F3 and B3) are annealed, hybridized and extended with a template to form a dumbbell-shaped intermediate product. The dumbbell-shaped intermediate product can be self-extended and can also be used as a template to be amplified by a primer, so that a large amount of stem-loop-like and vegetable-like amplification products are generated, and exponential amplification is initiated. In the accelerated version of LAMP, an accelerating primer is combined to a loop single chain of an amplification product stem-loop structure, and then extension and strand displacement reactions are carried out under the catalysis of Bst DNA polymerase, so that more stem-loop-like products are amplified and the reaction speed is accelerated. Specific LAMP amplifications and their typical amplification products are shown in FIG. 3: the positive control exhibits an "S-shaped" amplification curve and produces a large amount of ladder-like specific amplification products, whereas the positive control lacks an amplification curve and amplification products. However, when the primer design is not good enough, non-specific amplification easily occurs in LAMP negative control, as shown in FIG. 4.

However, LAMP has the following disadvantages: (1) LAMP primer design is complex, and when the DNA to be detected is short, an appropriate LAMP primer is difficult to design. (2) The LAMP amplification requires a large number of primers, and non-preferred primers are likely to cause non-specific amplification.

Disclosure of Invention

The technical scheme provides a novel isothermal amplification method named Stem-loop-primer-assisted isothermal nucleic acid amplification (SPA). The SPA has the advantages of simple operation, rapidness, sensitivity, economy and the like, can realize the rapid amplification and detection of nucleic acid, and is particularly suitable for field detection and rapid screening. The specific technical scheme is as follows:

a stem loop primer assisted isothermal nucleic acid amplification technology comprises the following steps:

a. SPA primer design:

two common primers, namely a Forward Primer (FP) and a reverse primer (BP), are designed for a template. Primer design was performed with reference to the PCR primer design rules.

In the step a, when designing the primer, in order to reduce the probability of forming primer dimer between FP and BP, the Δ G of primer dimer at the 3' end should be less than 2.0 kcal/mol, and the Tm values of FP and BP are 45-55 ℃, and the length of the amplification product of FP and BP is 40BP to 80 BP.

b. After designing the common primers, Stem-loop sequences are added to the 5' ends of FP and BP to form a pair of Stem-loop primers, namely a forward Stem-loop primer (SFP) and a reverse Stem-loop primer (SBP).

In the step b, the stem length of the stem-loop sequence is generally 6BP to 14 BP, a nucleic acid secondary structure should be avoided from being formed by the stem-loop sequence, FP and BP, and the nucleic acid secondary structure can be analyzed and predicted by online software MFold (http:// unaford.

c. Preparation of SPA primer mixed solution: the SPA amplification primer is formed by mixing common primers (FP and BP) and stem-loop primers (SFP and SBP).

In the step c, the proportion of the stem-loop primer in the primer mixture is different from 1% to 50%. The concentration of the mixed primers was 3-6. mu.M.

d. SPA buffer formulation (2 × SPA mix):

0.149 g of potassium chloride, 0.264 g of ammonium sulfate, 0.395 g of magnesium sulfate heptahydrate, 15g of betaine, 3.0 mL of Tris-HCl (1.5M, pH =8.5), 10.0 mL of 25 mM dNTP, 1.0 mL of 100-fold diluted SYBR green I, and 2.0 mL of Tween-20 were weighed, fully dissolved and made to 100 mL, and 2.0 mL of Tween-20 was dispensed per tube and stored at-20 ℃.

e. Preparation of SPA reaction liquid:

the SPA reaction is a 10-50 system, preferably a 25 muL system: the method comprises 12.5 muL of 2 xSPAMix, 0.5-1 muL of Bst DNA polymerase (8U/muL), 2-5 muL of primer mixture, a template to be detected and finally water supplementing to 25 muL. The reaction tube was incubated in a real-time fluorescent thermostat (55-65 ℃) for 60-120 minutes.

The amounts of the primer, the template, the enzyme and other components in the reaction, the reaction time and the reaction temperature are all adjusted according to the specific target to be detected and the experimental conditions.

The amplification principle of the present invention is shown in FIG. 5:

the invention firstly simplifies the design of the primers: in the LAMP technology in the prior art, 4 primers are designed at least aiming at 6 areas of a template, so that constant temperature amplification can be realized. The invention adopts a brand-new primer design principle, and is called a Stem-loop-primer-assisted novel isothermal nucleic acid amplification technology (SPA) for short. The SPA primer design is very simple, only need to design 1 pair of common primers aiming at 2 regions of the template, namely forward primer (Forwardprimer, FP) and reverse primer (Backwardrimer, BP). Then 1 pair of Stem-loop primers, namely a forward Stem-loop-forward-primer (SFP) and a reverse Stem-loop-backward-primer (SBP), are constructed on the basis of the common primers. The stem-loop primers SFP and SBP are derived from FP and BP, and only a section of universal stem-loop sequence is added at the 5' end of the stem-loop primers SFP and SBP. Therefore, the 3' -terminal sequences of SFP and SBP are completely identical to FP and BP, and also completely identical to the binding region of the template. The SPA amplification primer is formed by mixing a common primer and a stem-loop primer according to a certain proportion.

The invention reduces the occurrence probability of nonspecific amplification: isothermal amplification techniques are relatively prone to non-specific amplification, which is mainly due to primer dimers. As mentioned earlier in the LAMP technology, 4 or 6 primers are required for amplification (consisting of only 6 or 8 DNA fragments, respectively), and thus primer dimer formation and non-specific amplification are very likely. However, the SPA can realize isothermal amplification only by 4 primers, and the 4 primers are only composed of 2 template fragments, so that the formation chance of primer dimers is greatly reduced, and the nonspecific amplification of the SPA is reduced.

The technical scheme of the invention has the following beneficial effects:

the isothermal amplification technology is a nucleic acid amplification technology which reacts at a constant temperature, and has the characteristics of rapidness, sensitivity, economy, no need of a thermal cycler and the like, so that the defects of PCR are greatly overcome. The isothermal amplification technology is an ideal technology for realizing rapid detection and field detection of nucleic acid, and is also an important development direction in the field of molecular diagnosis. LAMP is a relatively widely used isothermal amplification technology, and has the obvious advantages of high sensitivity, high reaction speed and good economy. However, LAMP also has significant disadvantages: the design of the primers is complex, the number of the primers is large, and the amplification specificity is poor. Efficient LAMP amplification requires 6 primers designed for eight sections of a template, which not only makes primer design difficult, but also further causes stronger background amplification and poorer specificity of LAMP technology. The reason is that the primer pair is easily formed by a plurality of primers, the mismatching probability of the primer and background nucleic acid is increased, the primer pair is easily formed by a plurality of primers, the mismatching probability of the primer and the background nucleic acid is increased, and the non-specific amplification is caused.

Compared with LAMP constant temperature technology, the technical scheme SPA is a constant temperature amplification technology which is simple, feasible, sensitive, specific, economical and practical. The SPA primer is simple in design, and rapid and sensitive isothermal amplification of nucleic acid can be realized only by designing a pair of common primers and stem-loop primers, so that rapid detection of pathogenic microorganisms is realized. In view of its simplicity, rapidity, sensitivity, and economy, SPA will be expected to be an ideal portable molecular diagnostic technique. Therefore, the SPA technology has wide application prospect.

In addition, the market size in the field of molecular diagnostics is huge, and POCT is a future development direction of in vitro diagnostics. The visualized SPA constant-temperature amplification technology is a simple, convenient and economic molecular diagnosis technology, and is expected to play an important role in the fields of bedside detection, field detection and family detection. The visualized SPA constant temperature amplification technology can be used for detecting the new coronavirus and has wide application value in the whole molecular diagnosis field. Therefore, the technology has important clinical significance, wide application prospect and great social and economic benefits.

Drawings

FIG. 1 is a schematic diagram of primer design for the prior art LAMP of the present invention;

FIG. 2 is a schematic diagram of the amplification principle of the prior art LAMP of the present invention;

FIG. 3 is a gel electrophoresis analysis diagram of the specific amplification and amplification products of the LAMP of the prior art of the present invention;

FIG. 4 shows the non-specific amplification of the LAMP of the prior art;

FIG. 5 is a schematic diagram of the principle of amplification of SPAs of the present invention;

FIG. 6 shows the SPA-amplified ALDH2 gene fragment in example 1 of the present invention;

FIG. 7 is a schematic diagram of the enzymatic digestion analysis of the ALDH2 amplification product in example 1 of the present invention;

FIG. 8 shows the restriction enzyme digestion and electrophoresis analysis in example 1 of the present invention;

FIG. 9 is a SPA amplification product sequencing analysis of example 1 of the present invention;

FIG. 10 is a verification of the MDI self-extending function of example 1 of the present invention;

FIG. 11 is a schematic diagram of the SPA primer and LAMP primer in example 1 of the present invention;

FIG. 12 is a comparison of the amplification effects of SPA and LAMP in example 1 of the present invention;

FIG. 13 is a comparison of the products of the SPA and LAMP amplifications in example 1 of the invention;

FIG. 14 is the sensitivity of SPA in example 1 of the present invention;

FIG. 15 is the specificity of SPA in example 1 of the present invention;

FIG. 16 shows the detection of HPV16 DNA by SPA in example 2 of the present invention.

Detailed Description

Example 1:

a stem loop primer assisted isothermal nucleic acid amplification technology is implemented by the following steps:

acetaldehyde dehydrogenase 2 (ALDH 2) is a key enzyme in the alcohol and nitroglycerin metabolism process, and the activity of the enzyme is lost after ALDH2 mutation, so that alcohol and nitroglycerin metabolism is disturbed. Therefore, the detection of the ALDH2 gene has important clinical representation significance. This example is directed to ALDH2 gene fragments chemically synthesized as templates and having the following nucleic acid sequences: 5'-CCGGGAGTTGGGCGAGTACGGGCTGCAGGCATACACTGAAGTGAAAACTGTGAGTGTGG-3' (derived from the reference sequence GenBank: AH 002599.2).

a. SPA primer design:

first, two common primers (FP and BP) are designed for the target nucleic acid in the forward and reverse directions: common primer FP (5'-CCGGGAGTTGGGCGAG-3') and common primer BP (5'-CCACACTCACAGTTTTCAC-3').

In step a, the Tm values of FP and BP are 53 ℃ and 49 ℃, respectively, and the amplification product length of FP and BP is 59 BP.

b. After designing the common primers, adding stem-loop sequences at the 5' ends of FP and BP to form stem-loop primers (SFP and SBP): hairpin primer SFP (5'-tttatatatatataaaCCGGGAGTTGGGCGAG-3') and hairpin primer SBP (5'-cctatatatatataggCCACACTCACAGTTTTCAC-3').

In the step b, the stem length of the stem-loop sequence is 8 BP, so that the stem-loop sequence, FP and BP are prevented from forming a nucleic acid secondary structure.

In the step c, the proportion of the stem-loop primer in the primer mixture is 40%. The concentration of the mixed primers was 5. mu.M.

d. SPA buffer formulation (2 × SPA mix):

e. Preparation of SPA reaction liquid:

the SPA reaction generally adopts a 25 mu L system: including 12.5 μ L of 2 xSPAMix, 1 μ L Bst DNA polymerase, 4 μ L of primer mixture, 2 μ L of template (1 x 106copies/μ L), and finally water supplement to 25 μ L. A negative control was also set.

The following experimental verification was performed in this example:

the feasibility and the principle correctness of the SPA are verified through real-time fluorescence analysis, enzyme digestion-electrophoresis analysis and product sequencing analysis.

(1) Real-time fluorescence analysis of the SPA amplification process:

placing the prepared reaction solution in a real-time fluorescence PCR instrument for incubation at a constant temperature of 63 ℃ for 90 minutes, monitoring the fluorescence intensity change in the reaction process by real-time fluorescence, wherein the real-time fluorescence result of the SPA amplified ALDH2 gene is shown in figure 6, and the SPA can efficiently amplify ALDH2 gene.

(2) Enzyme digestion-electrophoresis analysis of SPA amplification products:

the ALDH2 gene fragment of this example contains PstI restriction enzyme sites as shown in FIG. 7, therefore, the amplification product is further subjected to enzyme digestion and electrophoretic analysis by PstI enzyme to observe the specificity of the amplification product and the correctness of the amplification mechanism. According to the reaction principle, the SPA amplification product of ALDH2 can be specifically digested by PstI enzyme, and four short fragments with different lengths are formed, as shown in FIG. 7. The enzyme digestion-electrophoresis analysis shows that the SPA amplification product is ladder-like DNA with different lengths, as shown in lane 1 of figure 8, and can be degraded into four short fragments with different lengths by PstI enzyme, as shown in lane 1 of figure 8. Thus, the specificity of the SPA amplification product and the correctness of the amplification mechanism are further demonstrated.

(3) Sequencing analysis of SPA amplification products:

to further observe whether the amplified product contains stem-loop sequence DNA, the shortest amplified product in lane 1 of FIG. 8 was recovered by cutting gel and then subjected to sequencing analysis. The sequencing result is shown in FIG. 9, and the shortest product fragment is a specific amplification product with stem-loop sequences at both ends, and we named it as a dumbbell intermediate (MDI) with unit length. The base sequence of MDI was completely identical to that of the original template (except for the stem-loop sequence), which further confirmed the specificity of the amplification product and the correctness of the amplification principle.

(4) The self-extension function of stem-loop sequence DNA is verified:

to further verify the self-extension function of the stem-loop sequence DNA, the shortest product fragment (MDI) in the SPA amplification product is cut and recovered, and then BstDNA polymerase is used for catalyzing MDI self-extension. The results show that BstDNA polymerase catalyzed MDI self-extension without primer addition, as shown in lane 3 of FIG. 10, to form longer amplification products. This further demonstrates the self-extension ability of the stem-loop sequence-bearing intermediate and the correctness of the SPA amplification mechanism.

The principle of SPA amplification was verified by the above experiments:

FIG. 6 shows the SPA amplified ALDH2 gene fragment of example 1; curve 1, positive control; curve 2, no template negative control; curve 3, no primer negative control;

FIG. 7 is a schematic diagram of the enzymatic digestion analysis of the ALDH2 amplification product of example 1 of the present invention;

FIG. 8 shows the restriction enzyme digestion and electrophoresis analysis in example 1 of the present invention; lane 1, amplification product of curve 1 in figure 6; lane 1, the amplification product of curve 1 in figure 6 digested with Pst i; lane 2, amplification product of curve 2 in figure 6; lane 3, amplification product of curve 3 in figure 6; lane 4, PCR amplification products with SFP and SBP as primers; lane 4, PCR amplification product digested with Pst i;

FIG. 9 is a sequence analysis of SPA amplification products of example 1 of the present invention; the shortest fragment in lane 1 of FIG. 7 was recovered by cutting and analyzed by sequencing. The results show that the shortest fragment is a unit length primary product (MDI) with stem-loop sequences at both ends;

FIG. 10 is a verification of the MDI self-extending function of example 1 of the present invention; lane 3 (MDI and Bst enzyme only) shows many large molecular weight amplification products, indicating self-extension of MDI.

Nonspecific amplification of SPA is lower than LAMP:

SPA is typically characterized by simple primer design and low probability of non-specific amplification due to: compared with the classical LAMP isothermal amplification technology, the SPA is a simplified version thereof, which not only reduces the number of primers, but also fully utilizes the self-extension capability of the stem-loop structure. In the LAMP reaction, due to the large number of primers, primer dimer and primer-background mismatch are easily caused, and these non-specific pairs will be amplified in the subsequent amplification reaction, resulting in higher background amplification and non-specific amplification. The number of primers for SPA is relatively small, and thus it is expected that the occurrence of the above-mentioned phenomenon can be greatly reduced. In order to verify the effect and advantage of SPA reduction of non-specific amplification, closely related SPA primers and LAMP primers (FIG. 11) are designed on the ALDH2 gene, namely F2/B2 of the LAMP primers is completely consistent with FP/BP of the SPA primers. And the other LAMP primers are designed according to the design principle. Then, SPA and LAMP amplification were performed under the same conditions using the same amplification reaction buffer and enzyme. The amplification results are shown in FIG. 12: the negative control for SPA showed no significant non-specific amplification within 90 minutes, whereas the negative control for LAMP showed significant non-specific amplification. Further, the amplified products were analyzed by electrophoresis, as shown in FIG. 13, the negative control of SPA showed no significant non-specific amplified products, while the negative control of LAMP showed significant non-specific amplified products.

Comparison of nonspecific amplification of SPA and LAMP:

FIG. 12 shows comparison of the amplification effects of SPA and LAMP in example 1;

FIG. 13 is a comparison of the products of the SPA and LAMP amplifications in example 1 of the invention.

Sensitivity of SPA:

we diluted the ALDH2 gene fragment in 10-fold gradient and made it to 10-100000 copies/. mu.L. The sensitivity of detection of SPA was then analyzed using these gradient diluted samples as shown in FIG. 14. The results show that, in a 25ul reaction system, SPA can stably detect about 1X 102 copies of template molecules, i.e. the limit-of-detection (LOD) reaches 6.65 aM.

The sensitivity of the SPA is shown in FIG. 14.

Specificity of SPA:

in order to research the amplification specificity of SPA, we used Escherichia coli DNA, Human Papilloma Virus (HPV) DNA, Hepatitis B Virus (HBV) DNA, EB virus (EBV) DNA, Tubercle Bacillus (TB) DNA, Hepatitis C Virus (HCV) RNA and the like as templates and amplified SPA primers for amplifying ALDH2 gene segments. As shown in FIG. 15, the SPA primers for amplifying the ALDH2 gene fragment could not amplify the above nonspecific nucleic acids, i.e., SPA had good specificity.

FIG. 15 specificity of SPA. In the constant temperature amplification process of 90 minutes, the SPA primer for amplifying the ALDH2 gene segment has no cross reaction with E.coli DNA, HPV-DNA, HBV-DNA, EBV-DNA, TB-DNA, HCV-RNA and the like.

Example 2:

human Papillomavirus Type 16 (HPV 16) is obviously related to the occurrence and development of cervical cancer, and the HPV16 infection rate of a cervical cancer patient reaches 57%, so that HPV16 detection is one of important detection items for early screening of cervical cancer. This example is directed to a HPV 16L 1 gene fragment (HPV 16-DNA) whose template is chemically synthesized, and whose nucleic acid sequence is: 5'-CACTGTCTACTTGCCTCCTGTCCCAGTATCTAAGGTTGTAAGCACGGATG-3' (from reference sequence GenBank: K02718.1).

a. SPA primer design, as shown in FIG. 16A:

first, two common primers (FP and BP) are designed for the target nucleic acid in the forward and reverse directions: common primer FP (5'-CACTGTCTACTTGCCTCCT-3'), common primer BP (5'-CATCCGTGCTTACAACCTT-3').

In the step a, the Tm values of FP and BP are 51 ℃ and 50 ℃ respectively, and the amplification product lengths of FP and BP are 50 BP.

b. After designing the common primers, adding stem-loop sequences at the 5' ends of FP and BP to form stem-loop primers (SFP and SBP): hairpin primer SFP (5'-tttatatatatataaaCACTGTCTACTTGCCTCCT-3') and hairpin primer SBP (5'-tttatatatatataaaCATCCGTGCTTACAACCTT-3').

In the step c, the proportion of the stem-loop primer in the primer mixture is 30%. The concentration of the mixed primers was 5. mu.M.

d. SPA buffer formulation (2 × SPA mix):

e. Preparation of SPA reaction liquid:

the SPA reaction generally adopts a 25 mu L system: including 12.5 muL of 2 xSPAMix, 0.8 muL of Bst DNA polymerase, 4 muL of primer mixture, 2 muL of HPV16-DNA solution (1 x 106 copies/muL), and finally water supplement to 25 muL. A negative control was also set.

Referring to FIG. 16, SPA amplified HPV 16-DNA. (A) A template to be detected and an SPA primer. (B) Real-time fluorescence monitoring of the amplification process. (C) And (5) analyzing the amplification product by gel electrophoresis.

The following experiments were carried out in this example:

SPA amplification of HPV 16-DNA:

and placing the prepared reaction solution in a real-time fluorescent PCR instrument for incubation at a constant temperature of 63 ℃ for reaction for 90 minutes. And (3) real-time fluorescence monitoring the fluorescence intensity change in the reaction process so as to observe the change condition of the amplification product of the SPA. The real-time fluorescence results of SPA amplification for HPV16-DNA are shown in FIG. 16B, SPA did amplify HPV16-DNA efficiently, and the negative control did not amplify non-specifically within 90 min.

Electrophoretic analysis of SPA amplification products:

the amplification products were further analyzed by gel electrophoresis, and the results are shown in FIG. 16C: no non-specific amplification product appears in the negative control, and the amplification product in the positive control is a typical ladder-like electrophoresis band, and the position of the minimum band (84 bp) is consistent with the expectation. This indicates that SPA can specifically amplify HPV 16-DNA.

Example 3: a stem loop primer assisted isothermal nucleic acid amplification technology comprises the following steps:

a. SPA primer design:

In the step a, when designing the primer, in order to reduce the probability of forming primer dimer between FP and BP, the Δ G of primer dimer at the 3' end should be less than 2.0 kcal/mol, and the Tm values of FP and BP are 50 ℃, and the length of the amplification product of FP and BP is 40 BP.

In the step b, the stem length of the stem-loop sequence is 6BP, and the stem-loop sequence should be prevented from forming a nucleic acid secondary structure with FP and BP.

In the step c, the proportion of the stem-loop primer in the primer mixture is 2%. The concentration of the mixed primers was 3. mu.M.

d. SPA buffer formulation (2 × SPA mix):

e. Preparation of SPA reaction liquid:

the SPA reaction is preferably carried out in a 25 mu L system: the method comprises 12.5 muL of 2 xSPAMix, 0.5-1 muL of Bst DNA polymerase (8U/muL), 2-5 muL of primer mixture, a template to be detected and finally water supplementing to 25 muL. The reaction tubes were incubated in a real-time fluorescent thermostat (55 ℃) for 120 minutes.

Example 4: a stem loop primer assisted isothermal nucleic acid amplification technology comprises the following steps:

a. SPA primer design:

In the step a, when designing the primer, in order to reduce the probability of forming primer dimer between FP and BP, the Δ G of primer dimer at the 3' end should be less than 2.0 kcal/mol, and the Tm values of FP and BP are at 55 ℃, and the length of the amplification product of FP and BP is 80 BP.

In the step b, the stem of the stem-loop sequence is 14 BP, a nucleic acid secondary structure should be avoided from being formed by the stem-loop sequence, FP and BP, and the nucleic acid secondary structure can be analyzed and predicted by online software MFold (http:// unafleld. rna. albany. edu).

In the step c, the proportion of the stem-loop primer in the primer mixture is 48%. The concentration of the mixed primers was 6. mu.M.

d. SPA buffer formulation (2 × SPA mix):

e. Preparation of SPA reaction liquid:

the SPA reaction is preferably carried out in a 25 mu L system: the method comprises 12.5 muL of 2 xSPAMix, 0.5-1 muLBst DNA polymerase (8U/muL), 2-5 muL of primer mixture, a template to be detected and finally water supplementing to 25 muL. The reaction tubes were incubated in a real-time fluorescent thermostat (65 ℃) for 60 minutes.

Claims

1. A stem loop primer assisted isothermal nucleic acid amplification technology is characterized by comprising the following steps:

a. SPA primer design: firstly, designing two common primers, namely a forward primer FP and a reverse primer BP, aiming at a template;

b. after designing a common primer, adding stem-loop sequences at the 5' ends of FP and BP to form a pair of stem-loop primers, namely a forward stem-loop primer SFP and a reverse stem-loop primer SBP;

c. preparation of SPA primer mixed solution: the SPA amplification primer is formed by mixing common primers FP and BP and stem-loop primers SFP and SBP;

d. SPA buffer preparation 2 XSAEmix: weighing potassium chloride, ammonium sulfate, magnesium sulfate heptahydrate, betaine, Tris-HCl, dNTP, 1.0 mL of 100-time diluted SYBR green I and Tween-20, fully dissolving and fixing the volume, and subpackaging each tube at-20 ℃ for storage;

e. preparation of SPA reaction liquid: the SPA reaction is a 10-50 muL system.

2. The stem loop primer assisted isothermal nucleic acid amplification technique of claim 1, wherein: in the step a, the Delta G of the 3' end primer dimer is less than or equal to 2.0 kcal/mol, the Tm values of FP and BP are between 45 and 55 ℃, and the lengths of amplification products of FP and BP are between 40 and 80 BP.

3. The stem loop primer assisted isothermal nucleic acid amplification technique of claim 1, wherein: in the step b, the stem length of the stem-loop sequence is 6-14 bp.

4. The stem loop primer assisted isothermal nucleic acid amplification technique of claim 1, wherein: in the step c, the proportion of the stem-loop primer in the primer mixture is 1-50%, and the concentration of the mixed primer is 3-6 mu M.

5. The stem loop primer assisted isothermal nucleic acid amplification technique of claim 1, wherein: in the step d, 0.149 g of potassium chloride, 0.264 g of ammonium sulfate, 0.395 g of magnesium sulfate heptahydrate, 15g of betaine, 3.0 mL of 1.5M Tris-HCl with pH =8.5, 10.0 mL of 25 mM dNTP, 1.0 mL of 100-fold diluted SYBR green I and 2.0 mL of Tween-20 are weighed, fully dissolved and fixed to 100 mL, and each tube of 2.0 mL is subpackaged and stored at-20 ℃.

6. The stem loop primer assisted isothermal nucleic acid amplification technique of claim 1, wherein: in the step e, the SPA reaction is a 25 mu L system, and comprises 12.5 mu L of 2 xSPAMix, 0.5-1 mu L of 8U/mu L of Bst DNA polymerase, 2-5 mu L of primer mixture, a template to be detected, water is supplemented to 25 mu L at last, and the reaction tube is placed in a real-time fluorescence thermostat at the temperature of 55-65 ℃ for incubation for 60-120 minutes.